The Effect of The Release of Exogenous Nitric Oxide on The Responses of The Pregnant Human Myometrium To Oxytocin*

Abstract Currently there is insufficient evidence to support the routine administration of nitric oxide donors in the treatment of threatened preterm labor. An understanding of the role that nitric oxide plays in the management of threatened preterm labor may lead to more effective treatment and prevention. This is why the aim of our study was to examine the involvement of exogenous nitric oxide release in regulating responses of the human pregnant myometrium to oxytocin. Biopsies of human myometrial tissue during pregnancy were obtained from 8 pregnant women, aged 21-35 years. The responses of the specimens to oxytocin in the absence and presence of a DETA/NO were recorded under isometric conditions. Preincubation with exogenous nitric oxide significantly (p<0.001) attenuated the contractile response of the uterine strips to oxytocin in concentrations higher than 10-8 mol/L. The inhibition of nitric oxide synthesis alone or in combination with DETA/NO incubation did not significantly change the oxytocin contractile effect in the concentration-response curve. Moreover, there was no significant variation in the mean value for log EC50 for oxytocin between the group with oxytocin alone and other groups. We present evidence in support of the hypothesis that continuous nitric oxide supply to the human pregnant myometrium environment attenuates its response to oxytocin but only when endogenous production of nitric oxide is not impaired.


INTRODUCTION
Preterm labor is de ned as the occurrence of regular painful uterine contractions of su cient frequency and intensity to e ect the progressive e acement and dilation of the cervix culminating in delivery of a preterm infant before 37 weeks of pregnancy [1]. Increased uterine contractility at term and preterm labor results from several mechanisms, one of which is the activation and stimulation of the myometrium by increasing the concentration of oxytocin (OXT) [2]. erefore, uterine contractions are induced via the activation of phospholipase C and release of inositol 1,4,5-triphosphate, 1,2-diacylglycerol, and intracellular calcium [2].
OXT mediates its biological e ects by acting on speci c receptors (OXTR) [3] but to some extent it also activates the myometrium via the vasopressin V 1a receptor [4]. e pregnant uterus is one of the traditional targets of OXT and is one of the most potent uterotonic agents clinically used to induce labor [5].
Nitric oxide (NO) is believed to participate in maintaining relaxation of the myometrium quiescence throughout gestation [6]. erefore, decreased production of NO as well as decreased myometrial response in late pregnancy is supposed to promote labor [7,8]. Functional experiments in vitro have reported that nitric oxide (NO) donors attenuate myometrial contractility [9]. To stimulate a preeclampsia-like syndrome in rats and mice NO production was blocked by administering a NOS-inhibiting agent, which implied a potential decrease in NO production and NOS activity [10]. However, several researchers disagree as to the importance of NO in preeclampsia. Some of them have suggested the possible primary or secondary role of NO in the development of preeclampsia, but few of them have associated the dysfunction of NO synthesis with other metabolic disorders described in this condition [11].
At present, there is insu cient evidence to support the routine administration of NO donors to the treatment of threatened preterm labor. Nevertheless, several studies have demonstrated the bene cial role of nitric oxide agents, in particular glyceryl trinitrate and L-arginine in lowering blood pressure and improving uteroplacental blood ow velocities [12]. Much research still needs to be done. Understanding the role of NO in the management of threatened preterm labor may lead to more e ective treatment and prevention. erefore, the aim of our study was to investigate the contribution of exogenous NO in the regulation of responses of the human pregnant myometrium to OXT.

Human myometrial Ɵssue
is study was carried out in accordance with the principles of the World Medical Association's Declaration of Helsinki, the International Conference on Harmonisation Guideline for Good Clinical Practice, and the laws and regulations of Poland. Biopsies of human myometrial tissue during pregnancy were obtained at elective caesarean section operations from 8 pregnant women, aged 21-35 years. All the women, who had previously quali ed for caesarean section surgery, were fully informed about the nature and procedure of the study and gave their written consent to participation. e Bioethics Committee of the Medical University of Białystok had earlier approved its protocol (Opinion No. R-I-002/109/2010).
Sample processing and data acquisiƟon e biopsies were excised from the upper lip of the lower uterine segment, incision in the midline, i.e. the upper portion of the lower uterine segment. Immediately upon collection, the samples were placed in ice-cold Tyrode's solution and transferred to the laboratory where they were processed as previously described [13]. e strips were then mounted in an isolated organ bath containing 20 ml of Tyrode's solution thermostatically maintained at 37 o C, pH 7.4, and bubbled with carbogen (95%O 2 + 5%CO 2 ). e strips were le for an equilibration period of 1-2 hours, within which the passive tension was adjusted to 2 mN. A er equilibration, regular phasic contractions were achieved.
Myometrium activity was recorded by a force transducer with digital output (BIO-SYS-TECH, Bialystok, Poland) and with the DASYLab so ware unit (version 9.0; Laboratory Data Acquisition System, SuperLogics, Waltham, MA, USA). Before each experiment, the strips were activated by 80 mmol/L K + . Appropriate controls were run under similar experimental conditions in strips of uterus obtained from the same woman. Only one doseresponse curve was performed on each preparation.
Diethylenetriamine/nitric oxide (DETA/NO)a spontaneously releasing nitric oxide donor at the concentration of 10 -4 mol/L was added to the organ bath in a series of experiments. To estimate the prolonged involvement of NO, spontaneous contraction recording (20 min) and again a er 20 hours were run under similar organ bath conditions obtained from the same patient. In the current study, 20 min preincubation with L-arginine analog NG -nitro-L-arginine (L-NNA) (3·10 -4 mol/L) was performed to inhibit endogenous NO production [14].
Oxytocin (OXT) was added cumulatively to the organ chambers (range 10 -14 -10 -6 mol/L) at 10-minute intervals, and the e ects were recorded. On the basis of the results obtained, incubation with 10 -4 mol/L DETA/NO with or without previous NOS inhibition (by L-NNA) was performed. en the observation of the OXT e ects on uterine contractions was conducted during continuous decomposition of DETA/NO. e responses were quanti ed by the area under the curve (AUC). e AUC re ected the total quantity of changes in amplitude, frequency of active contractions, and basal tension of myometrial strips over time before and a er the administration of the given drug. e AUC was measured as the area under all recorded contractions over a 10-minute interval before the addition of the substances that were tested [15].
A stock solution of OXT was prepared daily using bidistilled water. A series of dilutions were prepared on the day of the experiment. All the substances were added directly to the organ bath containing a Tyrode solution composed of (mM): NaCl 136.9; KCl 2.70; MgCl 2 1.05; NaH 2 PO4 1.33; CaCl 2 1.80; NaHCO 3 25.0; and glucose 5.0. which was also made on a daily basis.

Measurements of contracƟon parameters
e contractile activity of the myometrial strips before the administration of the substances tested was treated as a control (set as 100%). e AUC was evaluated by calculating the integral of the appropriate section of the curve. Concentration-response curves were tted to the logistic equation using nonlinear regression (PRISM 6.0, Graph Pad So ware Inc., San Diego, CA, USA). e maximal response (E max ) was expressed as a percentage of the contractile activity before the administration of the substances tested, whereas the concentrations of agents that resulted in a half-maximal e ect were expressed as -log EC 50 .

StaƟsƟcal analysis
All the results were expressed as means ± SEM with "N" denoting the number of experiments performed on myometrial strips from di erent patients. Dose-response was determined using analysis of variance (ANOVA) followed by a non-parametric or parametric Dunnett's multiple comparison test where appropriate. All the analyses were performed using Prism 6 for Windows (version 6.0, GraphPad So ware Inc., San Diego, CA). Values were considered to be statistically signi cant at p<0.05.

RESULTS
Spontaneous phase contractile activity was developed in 75.00% of the myometrium strips obtained from pregnant women (Fig. 1). All the strips examined presented a similar basal tension, mean 1.97±0.82 mN (N=6). e mean amplitude was 4.68±0.42 mN and the mean frequency of spontaneous contractions for 10 min was 1.72±0.31.

Effects of LNNA
Inhibition of NOS by 3·10 -4 mol/L L-NNA caused an intensi cation of the spontaneous contractile activity of myometrial strips demonstrated as a signi cant increase of the AUC 115.5±2.11% (Fig. 1B). is e ect is similar to that shown in other tissues [14,16] and in like manner involved a signi cant increase in the basal tension, the mean frequency and amplitude of contractions.

Effects of DETA/NO
To examine the in uence of the prolonged e ects of exogenous NO release on uterine contractility, we observed it a er 20 min (Fig. 2) or 20-hour incubation with 10 -4 mol/L DETA/NO. DETA/NO has been shown to release nitric oxide with an estimated half-life of about 20 hours in a solution with pH 7.4 at 37° C [17]. In the current studies, this compound produced a substantial inhibition of spontaneous contractility a er 20 min and considerably stronger a er 20-hour incubation of myometrial strips (92.45±2.44% and 63.74±4.58%, respectively, p<0.001). A er 20-hour incubation, the AUC of the control pregnant strips changed signi cantly (91.54±2.52%, p<0.05). When we examined the e ect of 20-min incubation with DETA/NO a er inhibiting endogenous NO production, the decrease was 62.99±7.23% (p<0.01) of AUC (Fig. 2).
To evaluate the e ect of DETA/NO on the OXT-induced maximum response of myometrial smooth muscle, we conducted experiments without and a er incubation with L-NNA. One of the doubtless results of the tests was the signi cant inhibition of contractile activity of the strips that were used. e weakening of contractions was deeper a er 30 min than a er 10 min of incubation with DETA/NO (45.39%±7.78, N=7; 76.52%±7.55, N=6, respectively, p< 0.05). A similar trend, but insigni cant, occurred when NOS production was inhibited, a er 30 min of DETA/NO incubationic 31.19%±8.43, N=7 and a er 10 min 46.72%±6.79, N=7. e attenuation in AUC value was considerably deeper a er 10 min of preincubation with the NO donor, when NOS was additionally inhibited (p<0.05). No signi cant changes were observed a er 30 minutes of incubation.

ContracƟle responses to oxytocin
In the pregnant uterine strips, OXT (range 10 -14 -10 -6 mol/L) caused a dose-dependent increase of AUC, as shown previously [18,19] (Fig. 3). e presence of 10 -4 mol/L DETA/NO caused a signi cant attenuation in the contractile response to OXT at concentrations higher than10 -8 mol/L. Inhibition of NO synthesis alone or in combination with DETA/NO did not alter the OXT contractile e ect in the concentration-response curve (Fig. 3).
Moreover, no signi cant variation in the mean value for log EC 50 for OXT (range 10 -14 -10 -6 mol/L) was observed between the group with OXT alone and other groups.
E max values for OXT a er preincubation with NO donor were extensively lower than in the other three groups (p<0.05). In contrast, E max values for OXT alone did not signi cantly di er compared to other groups (Tab. I).

DISCUSSION
Inhibition of NOS signi cantly increased myometrial contractility, which was to be expected for tissue in which NOS activity is regulating muscle tension. is observation is consistent with the ndings of Buhimschi et al., who reported that incubation with L-NAME increased the tension of rat myometrial strips in a concentrationdependent manner. [16]. Our previous report on human tissue was also consistent with the current results [14]. Also in a clinical study, Maciejewski et al. demonstrated that the decreased level of NO in patients with preterm labor may be associated with the initiation of uterine contraction before 37 weeks of gestation [7]. However, the results presented are in marked contrast with the ndings of Bartlett et al. that eNOS, iNOS and nNOS proteins were not expressed at detectable levels in myocytes of the human myometrium at any stage of pregnancy. [20] Nitric oxide donors cause attenuation of the myometrial contractility in humans [14,16] as well as in animals [9,21], which was also con rmed in our present study. Stronger inhibition a er 20-hour incubation than a er 20 min incubation seems to contrast with the release rate of the NO  donor according to the decay law dx= -λxdt. It has been found that numerous substances that are administered are distributed and metabolized according to the exponential distribution [22]. Control pregnant uterine strips a er 20-hour incubation showed a substantial decrease in spontaneous contractions. Increased sensitivity to the stimuli of the pregnant uterus may cause its di erent response to NO prolonged exposure. Unpredicted outcome was our observation of a signi cant reduction in myometrium contractions a er 20-minute incubation with DETA/NO a er earlier inhibition of endogenous NO production by L-NNA compared to 20-minute incubation with DETA/NO alone. We anticipated to sum up the e ect of endogenous and exogenous NO in uence on uterine contractions. It has been con rmed, both by in vitro and in vivo studies that exogenous NO can decrease endogenous eNOS activity, regardless of the changes in gene expression [23]. However, our results demonstrated that when the functional balance between constrictor factors and endogenous NO is a ected, exogenous NO appears to have a stronger impact on the attenuation of the uterine contractility.
is e ect can be explained by the predominance of the cGMP-independent pathway over the cGMP-dependent cascade when NOS is inhibited. Moreover, this excessive e ect is much stronger [24]. It should also be taken into account that endogenously generated NO can have an inhibitory in uence on both cGMP-dependent or -independent smooth muscle relaxation evoked by exogenous NO donors.
It is extensively acknowledged that the augmented production of NO by the pregnant myometrium serves to maintain relative uterine quiescence and a decrease of NO production close to term may be one of the triggering signals to start delivery activity. Changes in iNOS or cGMP expression have been examined in pregnant rats, guinea pigs, rabbits, and humans. Taken together, these studies support the hypothesis that endogenously produced NO via iNOS maintains relative myometrial quiescence during pregnancy, although the role of NO in the human myometrium has been questioned [20]. OXT has long been ascribed a major involvement in the mechanisms of labor, both in preterm and at term pregnancies. e present study showed that the continuous release of NO from its donor substantially decreased the response of human pregnant uterine strips to increasing concentrations of OXT.
In recent years, it has been hypothesized that NOmediated relaxation is dependent on S-nitrosylation of speci c and critical proteins involved in the relaxation of uterine smooth muscle [25]. It has been reported that prolonged exposure to DETA/NO of some lines of human cell culture results in dinitrogen trioxide (N 2 O 3 ) formation and S-nitrosylation of key cellular proteins [26]. erefore, it is possible that this process may result in contraction when considering the myosin light chain phosphatase. DETA/NO mediated cytotoxicity, through the action of N 2 O 3 at high concentration or in the case of long-term exposure, can cause cell death. e above e ects should be tested in experiments using N 2 O 3 scavengers. On the other hand, the in uence of N 2 O 3 scavengers on smooth muscle contraction, as used by Ali et al., has not been tested, while for example morpholine (another N 2 O 3 scavenger) exhibits vasodilator activity in vitro and in vivo [26,27].
us, further research should be carried out to explain the mechanisms of both an additional DETA/NO inhibitory e ect on spontaneous contractility and increased contractile response to OXT.
Prolonged exposure to DETA/NO may activate the MAPK/ERK signaling system and increase the production of interleukin 6 (IL-6) in skeletal myotubes, and IL-6 is a potent pro-in ammatory cytokine in human myometrium. IL-6 increases cyclooxygenase-2 and OXTR in cultured uterine smooth muscle cells but IL-1β initiates pregnant uterine contractility [28]. IL-6 initially causes contraction and then reduces cellular metabolic function with prolonged exposure [29]. erefore, further immunohistochemical studies with blockers to the MAPK/ERK signaling system could explain both the additional inhibitory e ect of DETA/NO on spontaneous contractility and the enhanced contractile response to OXT.
Current functional experiments with previous data [9,16] support the possible application of NO donors as tocolytics in preterm labor. However, when NO synthesis was inhibited, the myometrium response to OXT did not change considerably, even in the presence of DETA/NO. is fact may indicate that the reaction of the pregnant myometrium to OXT changes only when endogenous NO is produced. In the animal model, administering the NOSinhibiting agent virtually produced all the symptoms of preeclampsia, suggesting a direct relationship between NO production and vascular changes in a normal pregnancy [10,30]. Interactions leading to abnormal physiology and clinical syndrome related to preeclampsia are very complex and diverse combinations of prompting factors can lead to an equivalent dysfunctional mechanism. In the light of the data presented, the use of NO donors in the treatment of preterm labor should be questioned and requires further investigation.
Undoubtedly, problems and questions, like many other previous studies on the pathophysiology of preeclampsia, still remain to be elucidated. Nevertheless, our study is part of the abundance of research on the basic science and physiological changes in this syndrome. Based on this research, we provide evidence to support the hypothesis that a continuous supply of NO to the human pregnant myometrium environment attenuates its response to OXT, but only when endogenous NO production is not impaired. Complicated NO-mediated physiology involving NO requires advanced biochemical and biomolecular research that can support or counter both our ndings and those reported above.